Product and process to regulate actin polymerization in T lymphocytes

ABSTRACT

The present invention relates to methods to regulate actin polymerization in T lymphocytes involved in tumorigenesis, inflammatory responses, immune responses, allergic responses and graft rejection responses, kits to perform such assays and methods to identify regulatory reagents that specifically control actin polymerization in T lymphocytes.

GOVERNMENT RIGHTS

[0001] This invention was made in part with government support underAI-30575A, AI-29903A and T-32A100048, all awarded by the NationalInstitutes of Health. The government has certain rights to thisinvention.

FIELD OF THE INVENTION

[0002] The present invention relates to a process for regulating actinpolymerization in T lymphocytes. The present invention also relates toassays and methods useful for identifying compounds that regulate actinpolymerization in a T lymphocyte.

BACKGROUND OF THE INVENTION

[0003] Mammalian cells have cytoskeletal networks that are associatedwith their plasma membrane. The cytoskeleton is comprised of a densenetwork of actin filaments and associated actin-binding proteins.Components of both the cytoskeletal network and the plasma membrane areimportant for cellular signalling by, for example, localizing andfocusing critical signalling molecules.

[0004] Certain mammalian cells comprise multichain surface receptorsthat enable a cell to respond to changes in the environment outside ofthe cell. One such multichain receptor is a T cell receptor (TCR)located on the surface of T lymphocytes. A TCR is a multichain,heteromeric structure composed of an antigen binding domain comprising αand β chains, and non-covalently associated signal transducingcomplexes, including CD3-γ, δ and ε chains, and the ζ chains. Signaltransduction events produced by TCR ligation with majorhistocompatibility complexes (MHC) induce a variety of cytoplasmicmetabolic changes. For example, gene transcription and production ofinterleukin-2 (IL-2) are promoted by TCR ligation with MHC molecules.

[0005] Abnormalities in T lymphocyte function can arise throughderegulation of signalling in T cells. Such diseases include, forexample, autoimmune diseases, immunodeficiency diseases andimmunoproliferative diseases. T lymphocyte function also contributes tograft rejection. To develop compounds that regulate the activity ofmolecules involved in T cell function, there must be an understanding ofthe molecules and interactions involved in such T cell related diseases.

[0006] Prior investigators have suggested that ligand binding convertssurface immunoglobulin (Ig) to a detergent insoluble form, and that Igreceptors subsequently undergo extensive degradation accompanied by theappearance of a detergent soluble membrane product (Braun et al., J.Immunol. 128:1198-1204, 1982). Parsey et al. (J. Immunol. 151:1881-1893,1993) hypothesize about a connection between actin polymerization andthe ability of immobilized anti-CD3 antibodies to stimulate changes incell shape and F-actin morphology. Furthermore, the expression of foursrc-family genes associated with T cell activation was shown to bespecifically blocked by cyclosporine (Furue et al., J. Immunol.144(2):736-739, 1990). Prior investigators, however, have failed toteach or appreciate that actin polymerization in T lymphocytes isspecifically regulated by the presence of a particular motif (i.e., animmunoreceptor tyrosine-based activation motif; ITAM) of a ζ chain or εchain of a TCR.

[0007] Although therapeutics exist that regulate immune activity in ananimal, problems have arisen due to the non-specific nature and harmfulside effects of such drugs. Despite a long-felt need to discovercompounds that specifically regulate T cell activity with minimalside-effects, the complexity and lack of understanding of signaltransduction networks in a T cell has hindered the development of suchcompounds. The present invention offers a method and product thatpermits regulation of specific steps of a signal transduction pathway incells having ITAM-containing receptors.

SUMMARY OF THE INVENTION

[0008] Despite the complexity of signal transduction networks in cells,the present invention provides for a method to regulate actinpolymerization in T lymphocytes by controlling the step in thesignalling pathway in which an ITAM of the ζ or ε chain of a TCRinteracts with a tyrosine kinase and/or an adaptor molecule. Theadvantages arising from this invention include the specific regulationof a step in a T lymphocyte signalling pathway that can regulate Tlymphocyte growth, differentiation, homing, proliferation and death. Thepresent inventors are the first to appreciate the specific molecularinteractions involved in the actin polymerization steps within a Tlymphocyte, and thus, are the first to propose a method and product thattargets this particularly important event within a T lymphocyte.

[0009] One aspect of the present invention includes a method to identifycompounds capable of regulating actin polymerization in a T lymphocyte,comprising: (a) contacting a putative regulatory compound with a Tlymphocyte having a T cell receptor chain selected from the groupconsisting of a zeta chain and an epsilon chain, to form a contactedlymphocyte; (b) combining the contacted lymphocyte with a moleculecapable of inducing the phosphorylation of the zeta chain or the epsilonchain; and (c) assessing the ability of the putative regulatory compoundto regulate actin polymerization in the lymphocyte. In particular, themethod further comprising assessing the amount of interleukin-2 producedby the lymphocyte.

[0010] Another aspect of the present invention includes a method toregulate actin polymerization in a T lymphocyte, comprising contacting aT lymphocyte with an effective amount of a regulatory reagent that iscapable of altering the activity of an immunoreceptor tyrosine-basedactivation motif (ITAM) of a ζ chain of a T cell receptor. A preferredITAM to regulate using the present method comprises the amino acidsequence SEQ ID NO:1. Regulation of actin polymerization by the presentmethod preferably alters a T lymphocyte function including growth,differentiation, homing, proliferation, apoptosis and anergy.

[0011] The present invention also includes a method to regulate actinpolymerization in a T lymphocyte, comprising contacting a T lymphocytewith an effective amount of a regulatory reagent that alters theactivity of an immunoreceptor tyrosine-based activation motif of an εchain of a T cell receptor. A preferred ITAM to regulate using thepresent method comprises the amino acid sequence SEQ ID NO:2.

[0012] One embodiment of the present invention includes a cellularsystem where a src-family tyrosine kinase is contacted with a T cellreceptor chain selected from the group consisting of a ζ chain and an εchain that is regulated by said src-family tyrosine kinase, theimprovement comprising regulating actin polymerization by contacting a Tlymphocyte with a reagent capable of binding to a protein including athird ITAM of a ζ chain, an ITAM of an ε chain and an SH2 domain.

[0013] Another embodiment of the present invention includes aformulation capable of regulating actin polymerization in a Tlymphocyte, the formulation comprising: (a) a regulatory reagent thatalters the activity of a molecule including an immunoreceptortyrosine-based activation motif of a ζ chain of a T cell receptor and animmunoreceptor tyrosine-based activation motif of a ε chain of a T cellreceptor in a cell; and (b) a pharmaceutically acceptable carrier.

[0014] The present invention also includes a kit to identify compoundscapable of regulating actin polymerization in a T lymphocyte, the kitcomprising: (a) a cell comprising a T cell receptor chain selected fromthe group consisting of a ζ chain, an ε chain, and actin monomers; and(b) a means for detecting the polymerization of the actin monomers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 illustrates the structure of CD25/ζ, CD25/ζY153F and CD8/εchimeric molecules.

[0016]FIG. 2 illustrates the predicted structures of truncated TCR-ζpolypeptides, CD25/ζ and CD8/ε chimeric molecules.

[0017]FIG. 3 illustrates flow cytometric profiles of mouse thymocyteslabelled with fluorescent TCR specific antibodies.

[0018]FIG. 4 illustrates IL-2 production by cells expressing CD8/ζ orCD25/ζ proteins in response to stimulation with antibodies specific forCD8-α or CD25.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to a method for regulating actinpolymerization in a T lymphocyte (i.e., T cell). Actin polymerizationrefers to the polymerization of actin monomers to form actin filaments.Actin filaments are one element of a cellular cytoskeleton. As usedherein, the term “cytoskeleton” refers to a structure comprising proteinfibers, including microfilaments comprising actin, microtubules and/orintermediate filaments. Particular structures of a cytoskeleton includestress fibers, and focal adhesions or adhesion plaques. A description ofcellular cytoskeletons, and in particular actin filaments, can be foundin Darnell et al. (Molecular Cell Biology, Scientific American Books,1990, which is incorporated herein by reference in its entirety).

[0020] The polymerization and depolymerization of cytoskeletal filamentscan be regulated by molecules involved in a signal transduction pathwayin a cell. As used herein, the phrase “signal transduction pathway”refers to at least one biochemical reaction, but more commonly a seriesof biochemical reactions, which result from interaction of a cell with astimulatory molecule. The interaction of a stimulatory molecule with acell generates a “signal” that is transmitted through a signaltransduction pathway, ultimately resulting in actin polymerization.

[0021] A signal transduction pathway of the present invention caninvolve a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of the cell. As used herein, the term “molecule” refers to aprotein, a lipid, a nucleic acid or an ion, and at times is usedinterchangeably with such terms. In particular, a signal transductionmolecule refers to a protein, a lipid, a nucleotide, or an ion involvedin a signal transduction pathway. Signal transduction molecules of thepresent invention include, for example, cell surface receptors andintracellular signal transduction molecules. As used herein, the phrase“cell surface receptor” includes molecules and complexes of moleculescapable of receiving a signal and the transmission of such a signalacross the plasma membrane of a cell. The phrase “intracellular signaltransduction molecule,” as used herein, includes those molecules orcomplexes of molecules involved in transmitting a signal from the plasmamembrane of a cell through the cytoplasm of the cell, and in someinstances, into the cell's nucleus. The phrase “stimulatory molecule”,as used herein, includes ligands capable of binding to cell surfacereceptors to initiate a signal transduction pathway, as well asintracellular initiator molecules capable of initiating a signaltransduction pathway from inside a cell.

[0022] One aspect of the present invention includes a method to regulateactin polymerization in a cell by controlling the activity of animmunoreceptor tyrosine-based activation motif (ITAM) contained in aprotein expressed in such cell. Such ITAM's can be contained in avariety of proteins, in particular receptors that comprise multipleproteins referred to as multisubunit immune recognition receptors(MIRRs). MIRRs include receptors having multiple noncovalentlyassociated subunits and are capable of interacting with tyrosinekinases. MIRRs can include, but are not limited to, T cell antigenreceptors, B cell antigen receptors, Fc receptors and CD22. One exampleof an MIRR is a T cell receptor (TCR) on the surface of a T lymphocyte(used interchangeably herein with the term T cell). A TCR as referred toherein includes a multichain, heteromeric structure consisting of anantigen binding domain comprising an alpha (α) and a beta (β) chain, andnon-covalently associated signal transducing complexes, CD3 and zeta (ζ)chains. TCRs are capable of binding to a ligand (as described in detailbelow) and are capable of initiating a signal transduction pathway in acell upon ligand binding. A TCR typically includes an external portionlocated on the outer surface of a plasma membrane of a cell, atransmembrane portion that spans the plasma membrane, and a cytoplasmicportion located on the inner surface of the plasma membrane.

[0023] A suitable ITAM amino acid motif that can be regulated using amethod of the present invention includes tyrosine, leucine and/orisoleucine residues having a spatial arrangement represented by anYXXLXXXXXXXYXXΨ amino acid motif, wherein X can be any amino acid and“Ψ” can be either leucine or isoleucine. A preferred ITAM that can beregulated using a method of the present invention includes the aminoacid sequence E-R-R-R-G-K-G-H-D-G-L-Y-Q-G-L-S-T-A-T-K-D-T-Y-D-A-L (SEQID NO:1), N-K-E-R-P-P-P-V-P-N-P-D-Y-E-P-I-R-K-G-Q-R-D-L-Y-S-G-L (SEQ IDNO:2), E-T-A-A-N-L-Q-D-P-N-Q-L-Y-N-E-L-N-L-G-R-R-E-E-Y-D-V-L (SEQ IDNO:3), K-Q-Q-R-R-R-N-P-Q-E-G-V-Y-N-A-L-Q-K-D-K-M-A-E-A-Y-S-E-I (SEQ IDNO:4), E-R-R-R-G-K-G-H-D-G-L-Y-D-S-H-F-Q-A-V-Q-F-G-N-R-R-E-R-E (SEQ IDNO:5), D-K-Q-T-L-L-Q-N-E-Q-L-Y-Q-P-L-K-D-R-E-Y-D-Q-Y-S-H-L (SEQ IDNO:6), E-V-Q-A-L-L-K-N-E-Q-L-Y-Q-P-L-R-D-R-E-D-T-Q-Y-S-R-L (SEQ IDNO:7), A-A-I-A-S-R-E-K-A-D-A-V-Y-T-G-L-N-T-R-N-Q-E-T-Y-E-T-L (SEQ IDNO:8), E-L-E-S-K-K-V-P-D-D-R-L-Y-E-E-L-N-H-V-Y-S-P-I-Y-S-E-L (SEQ IDNO:9), E-T-N-N-D-Y-E-T-A-D-G-G-Y-M-T-L-N-P-R-A-P-T-D-D-D-K-N-I-Y-L-T-L(SEQ ID NO:10), D-M-P-D-D-Y-E-D-E-N-L-Y-E-G-L-N-L-D-D-C-S-M-Y-E-D-I (SEQID NO:11), D-A-G-D-E-Y-E-D-E-N-L-Y-E-G-L-N-L-D-D-C-S-M-Y-E-D-I (SEQ IDNO:12), D-G-K-A-G-M-E-E-D-H-T-Y-E-G-L-N-I-D-Q-T-A-T-Y-E-D-I (SEQ IDNO:13), D-S-K-A-G-M-E-E-D-H-T-Y-E-G-L-D-I-D-Q-T-A-T-Y-E-D-I (SEQ IDNO:14), D-R-Q-N-L-I-A-N-D-Q-L-Y-Q-P-L-G-E-R-N-D-G-Q-Y-S-Q-L (SEQ IDNO:15), P-E-I-S-L-T-P-K-P-D-S-D-Y-Q-A-L-L-P-S-A-P-E-I-Y-S-H-L (SEQ IDNO:16), D-Y-Q-A-L-L-P-S-A-P-E-I-Y-S-H-L-S-P-V-K-P-D-Y-I-N-L (SEQ IDNO:17), D-P-Y-W-G-N-G-D-R-H-S-D-Y-Q-P-L-G-T-Q-D-Q-S-L-Y-L-G-L (SEQ IDNO:18) and M-P-T-F-Y-L-A-L-H-G-G-Q-T-Y-H-L-I (SEQ ID NO:19), with anITAM having the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2 beingeven more preferred.

[0024] The term “activity” refers to any stage of activation of a signaltransduction molecule by, for example, binding of a target or substratemolecule, a conformational change of a molecule which results in theacquisition of catalytic activity by the molecule; the phosphorylationof a molecule, thereby resulting in the acquisition or loss of catalyticactivity by the molecule; or the translocation of a molecule from oneregion of a cell to another, thereby enabling the molecule to bindanother molecule. The term “regulate” refers to controlling the activityof a molecule and/or biological function, such as enhancing ordiminishing such activity or function.

[0025] A suitable cell useful in the present method includes any cellcomprising a protein having an ITAM. Cells for use with the presentinvention include mammalian, invertebrate, fungal, yeast and bacterialcells. Preferably cells for use with the present invention includemammalian cells and more preferably human cells. Particularly preferredcells for use with the present method include T cells and B lymphocytes(B cells).

[0026] One embodiment of the present invention is a method to regulateactin polymerization in a T cell, comprising contacting a T cell with aneffective amount of a regulatory reagent that is capable of altering theactivity of an ITAM of a zeta (ζ) chain of a TCR. Another embodiment ofthe present invention is a method to regulate actin polymerization in aT cell, comprising contacting a T cell with an effective amount of aregulatory reagent that alters the activity of an ITAM of an epsilon (ε)chain of a TCR. According to the present invention, altering theactivity of an ITAM includes either enhancing or reducing the activityof the ITAM depending upon the desired effect. For example, if thepresent method is employed to treat immunodeficiency or tumor anergy,then the method is performed to enhance ITAM activity. If the presentmethod is employed to treat graft rejection, then the method isperformed to reduce ITAM activity. Suitable cells for use with thepresent invention include any cell that has a ζ chain or an ε chain in anative physiological context (e.g., mature T cells, immature T cells, Tlymphomas, leukemias, natural killer cells, natural killer lymphomas andnatural killer leukemias). A ζ chain refers to the ζ subunit of a TCRcomplex (described in detail below). An ε chain refers to an ε subunitfrom a CD3 complex. A CD3 complex consists of γ, δ and ε chains.

[0027] According to the present invention, the present method is usefulfor the regulation of actin polymerization in mature and immature Tcells. A mature T cell is defined as a T cell having either CD4 or CD8expression in conjunction with high levels of TCR expression. Animmature T Cell can be a thymocyte at any stage prior to formation of amature thymocyte (e.g., a T cell that does not express either CD4 or CD8known as a double-negative cell; or a T cell that expresses both CD4 andCD8 known as a double-positive cell).

[0028] Effective amounts of a regulatory reagent can comprise an amountthat regulates actin polymerization to an extent such that a biologicalfunction of a cell that is controlled by actin polymerization ismodified. For example, an effective amount can comprise an amount thatprevents actin polymerization an extent that a T cell no longer secretesinterleukin-2 (IL-2). The amount of the regulatory agent can varydepending upon the type of regulatory reagent being administered to thecell and the type of cell. For example, the ease with which theregulatory reagent can cross the plasma membrane of a cell will dictatethe effective concentration of the reagent (e.g., more reagent beingnecessary if transport across a membrane is impaired, etc.).

[0029] A suitable regulatory reagent, or a mimetope thereof, of thepresent invention is capable of altering the activity of an ITAM of aprotein. In particular, a regulatory reagent, or a mimetope thereof, iscapable of regulating T lymphocyte function, including growth,differentiation, proliferation, apoptosis, anergy and/or homing, morepreferably IL-2 production. As used herein, anergy refers to thediminished reactivity by a T cell to an antigen and apoptosis refers tocell death. As used herein, homing refers to the movement of a Tlymphocyte in response to a molecule. For example, a T cell can home toa site of inflammation in response to molecules secreted by cellsinvolved in the inflammatory response.

[0030] Preferably, a regulatory reagent, or a mimetope thereof, iscapable of regulating the activity of an ITAM by, for example, alteringthe interaction between an ITAM and its substrate; altering the bindingbetween an ITAM and its target molecule; or altering the enzymaticactivity of a target molecule that binds to an ITAM and phosphorylatesthe ITAM. As used herein, “altering the binding” can refer to alteringthe affinity of one molecule for another, blocking the situs of bindingbetween two molecules, or interfering with the delivery of a molecule tothe area of another molecule or allosterically altering a molecule sothat it has either enhanced or diminished binding abilities. A “targetmolecule” refers to a molecule that can activate a signal transductionmolecule by binding to the signal transduction molecule. A “substratemolecule” refers to a molecule acted upon by a signal transductionmolecule. More preferably, a regulatory reagent, or mimetope thereof,binds to-an ITAM; to a src homology region 2 (SH2) domain of asrc-family kinase, syk-family kinase, adaptor molecule, PI-3 kinase or a14-3-3 protein; to a src homology 3 (SH3) domain specific forproline-rich sequences or non-proline-rich sequences; to oligoprolinecontaining actin binding proteins; to other actin binding proteinscontaining SH2 and/or SH3 domains; to plextrin homology domains (PH); toGLGF domains; or to WD domains.

[0031] A suitable regulatory reagent of the present invention includes afull or partial protein-based compound, a carbohydrate-based compound, alipid-based compound, a nucleic acid-based compound, a natural organiccompound, a synthetically derived organic compound or an antibody. Apreferred regulatory reagent includes a peptide, a polypeptide or anantibody.

[0032] A preferred peptide, or a mimetope thereof, of the presentinvention comprises an ITAM amino acid motif as described in detailherein and/or an SH2 domain. A more preferred peptide, or a mimetopethereof, of the present invention comprises an SH2 domain of a protein,including Fyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck,fak-B, PI-3 kinase and 14-3-3, with an SH2 domain of a protein,including Fyn, Lck, Zap-70, Shc, Syk, fak-B and 14-3-3 being morepreferred.

[0033] Particularly preferred peptides, or mimetopes thereof, of thepresent invention include sequences comprising at least a portion of anSH2 amino acid sequence described in Pelliuci et al. (Cell 70:93-104,1992), Chan et al. (Cell 71:649-662, 1992), Olivier et al. (Cell73:179-191, 1993), Taniguchi et al. (J. Biol. Chem. 266:15790-15794,1991), Sabe et al. (Proc. Natl. Acad. Sci. USA 89:2190-2194, 1992) andKoch et al. (Science 252:668-673, 1991).

[0034] In another embodiment, a regulatory reagent of the presentinvention is a peptide, or a mimetope thereof, of the present inventionas described herein that is phosphorylated. Preferably, a peptide isphosphorylated on a tyrosine residue. Examples of phosphorylatedpeptides of the present invention, in which “Y(P)” represents aphosphorylated tyrosine residue, includeE-R-R-R-G-K-G-H-D-G-L-Y(P)-Q-G-L-S-T-A-T-K-D-T-Y-D-A-L (SEQ ID NO:20)E-R-R-R-G-K-G-H-D-G-L-Y-Q-G-L-S-T-A-T-K-D-T-Y(P)-D-A-L (SEQ ID NO:21),E-R-R-R-G-K-G-H-D-G-L-Y(P)-Q-G-L-S-T-A-T-K-D-T-Y(P)-D-A-L (SEQ IDNO:22), N-K-E-R-P-P-P-V-P-N-P-D-Y(P)-E-P-I-R-K-G-Q-R-D-L-Y-S-G-L (SEQ IDNO:23), N-K-E-R-P-P-P-V-P-N-P-D-Y-E-P-I-R-K-G-Q-R-D-L-Y(P)-S-G-L (SEQ IDNO:24) and N-K-E-R-P-P-P-V-P-N-P-D-Y(P)-E-P-I-R-K-G-Q-R-D-L-Y(P)-S-G-L(SEQ ID NO:25).

[0035] In one embodiment, a regulatory reagent of the present inventionincludes an antibody that binds specifically to a signal transductionmolecule in such a manner that the activity of the molecule is altered.A preferred antibody useful as a regulatory reagent of the presentinvention binds specifically to a protein, including but not limited toFyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck, fak-B, PI-3kinase or 14-3-3. Another preferred antibody useful as a regulatoryreagent of the present invention binds specifically to a proteinincluding, for example, proteins that sequester actin monomers (e.g.,profilin); proteins that control nucleation of an actin polymer (e.g.,villin); proteins that block the barbed end of an actin polymer (e.g.,fragmin); proteins that block the pointed end of an actin polymer (e.g.,β-actin); proteins that sever an actin filament (e.g., gelsolin);proteins that depolymerize an actin polymer (e.g., depactin); or focaladhesion kinase, paxillin, tensin, annexin, ezrin, clathrin-H chain,vinculin, talin, zixin, cortactin, AFAP-110, p120, β catenin, connexin43and cadherins.

[0036] In accordance with the present invention, a “mimetope” refers toany compound that is able to mimic the ability of a regulatory reagentof the present invention. A mimetope can be a peptide that has beenmodified to decrease its susceptibility to degradation but that stillretains regulatory activity. Other examples of mimetopes include, butare not limited to, protein-based compounds, carbohydrate-basedcompounds, lipid-based compounds, nucleic acid-based compounds, naturalorganic compounds, synthetically derived organic compounds,anti-idiotypic antibodies and/or catalytic antibodies, or fragmentsthereof having desired regulatory activity. A mimetope can be obtainedby, for example, screening libraries of natural and synthetic compoundsfor compounds capable of regulating actin polymerization in a T cell, asdisclosed herein. A mimetope can also be obtained by, for example,rational drug design. In a rational drug design procedure, thethree-dimensional structure of a compound of the present invention canbe analyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. The three-dimensional structure can then be used topredict structures of potential mimetopes by, for example, computermodelling. The predicted mimetope structures can then be produced by,for example, chemical synthesis, recombinant DNA technology, or byisolating a mimetope from a natural source (e.g., plants, animals,bacteria and fungi).

[0037] Another aspect of the present invention includes a formulationcomprising a regulatory reagent of the present invention and apharmaceutically acceptable carrier. As used herein, the-term “a” canrefer to at least one (i.e., one or more). A formulation of the presentinvention can include a regulatory reagent that is capable of regulatingactin polymerization in a T cell, resulting in regulation of T cellfunction. Preferably, a formulation of the present invention comprises acombination of one or more peptides as described herein, or mimetopesthereof; a combination of antibodies as described herein, or mimetopesthereof; or a combination of antibodies and peptides as describedherein, or mimetopes thereof.

[0038] As used herein, a “pharmaceutically acceptable carrier” refers toany substance suitable as a vehicle for delivering a regulatory reagentof the present invention to a suitable in vitro or in vivo site ofaction. As such, carriers can act as a pharmaceutically acceptableexcipient or formulation of a therapeutic composition containing aregulatory reagent of the present invention. Preferred carriers arecapable of maintaining a regulatory reagent of the present invention ina form that is capable of altering signal transduction in a cell.Examples of such carriers include, but are not limited to water,phosphate buffered saline, Ringer's solution, dextrose solution,serum-containing solutions, Hank's solution and other aqueousphysiologically balanced solutions. Aqueous carriers can also containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity. Suitable auxiliary substances include, forexample, sodium acetate, sodium chloride, sodium lactate, potassiumchloride, calcium chloride, and other substances used to producephosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliarysubstances can also include preservatives, such as thimerosal, m- ando-cresol, formalin and benzol alcohol. Preferred auxiliary substancesfor aerosol delivery include surfactant substances non-toxic to arecipient, for example, esters or partial esters of fatty acidscontaining from about six to about twenty-two carbon atoms. Examples ofesters include, caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric, and oleic acids. Formulations of the presentinvention can be sterilized by conventional methods and/or lyophilized.

[0039] Useful carriers for a regulatory reagent of the present inventioninclude any artificial or natural lipid-containing target molecule,preferably cells, cellular membranes, liposomes, and micelles.Preferably, formulations of the present invention are administered inthe form of liposomes or micelles. Liposome and micelles of the presentinvention are capable of delivering a regulatory reagent from theextracellular space of a cell to the intracellular space of a cell.Concentrations of a regulatory reagent of the present invention combinedwith a liposome or a micelle include concentrations effective fordelivering a sufficient amount of the regulatory reagent to a cell suchthat signal transduction in such cell is regulated.

[0040] A formulation of the present invention comprises at least one ofthe regulatory reagents of the present invention as described above andmay also include at least one additional compound capable of regulatingsignal transduction. In one embodiment, a formulation of the presentinvention includes at least one isolated ITAM peptide and any other drugused for blocking actin polymerization or depolymerizing actin polymersin a cell. Examples of compounds capable of disrupting actinpolymerization include cytochalasins; proteins that sequester actinmonomers (e.g., profilin); proteins that control nucleation of an actinpolymer (e.g., villin); proteins that block the barbed end of an actinpolymer (e.g., fragmin); proteins that block the pointed end of an actinpolymer (e.g., β-actin); proteins that sever an actin filament (e.g.,gelsolin); proteins that depolymerize an actin polymer (e.g., depactin);and compounds that regulate the activity of a protein including, focaladhesion kinase, paxillin, tensin, annexin, ezrin, clathrin-H chain,vinculin, talin, zixin, cortactin, AFAP-110, p120, β catenin, connexin43and cadherins.

[0041] It is within the scope of the invention that isolated nucleicacid molecules that encode a regulatory reagent of the present inventionas herein disclosed can be used to produce such reagents. Methods tocreate and use such nucleic acid molecules are known to those of skillin the art. For example, a nucleic acid molecule encoding a peptide ofthe present invention can be chemically synthesized based on the aminoacid sequence of the peptide, ligated into an expression vector andtransformed into cells to produce a desired peptide.

[0042] A nucleic acid molecule as described herein can be DNA, RNA, orhybrids or derivatives of either DNA or RNA. Nucleic acid molecules asreferred to herein can include regulatory regions that controlexpression of the nucleic acid molecule (e.g., transcription ortranslation control regions), full-length or partial coding regions, andcombinations thereof. It is to be understood that any portion of anucleic acid molecule can be produced by: (1) isolating the moleculefrom its natural milieu; (2) using recombinant DNA technology (e.g., PCRamplification, cloning); or (3) using chemical synthesis methods. A geneincludes all nucleic acid sequences related to a signalling molecule,such as regulatory regions that control production of a cell surfacereceptor encoded by that gene (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself.

[0043] A nucleic acid molecule can include functional equivalents ofnatural nucleic acid molecules encoding a protein. Functionalequivalents of natural nucleic acid molecules can include, but are notlimited to, natural allelic variants and modified nucleic acid moleculesin which nucleotides have been inserted, deleted, substituted, and/orinverted in such a manner that such modifications do not substantiallyinterfere with the nucleic acid molecule's ability to encode a moleculeof the present invention. Preferred functional equivalents includesequences capable of hybridizing under stringent conditions (i.e.,sequences having at least about 70% identity), to at least a portion ofa signal transduction protein encoding nucleic acid molecule accordingto conditions described in Sambrook et al., ibid.

[0044] As guidance in determining what particular modifications can bemade to any particular nucleic acid molecule, one of skill in the artshould consider several factors that, without the need for undueexperimentation, permit a skilled artisan to appreciate workableembodiments of the present invention. For example, such factors includemodifications to nucleic acid molecules done in a manner so as tomaintain particular functional regions of the encoded proteinsincluding, a ligand binding site, a target binding site, a kinasecatalytic domain, etc. Functional tests for these variouscharacteristics (e.g., ligand binding studies and signal transductionassays such as kinase assays, and other assays described in detailherein and those known by those in the art) allows one of skill in theart to determine what modifications to nucleic acid sequences would beappropriate and which would not.

[0045] Transformation of a heterologous nucleic acid molecule (e.g., aheterologous cell surface receptor encoding a nucleic acid molecule)into a cell suitable for use in the present invention can beaccomplished by any method by which a gene is inserted into a cell.Transformation techniques include, but are not limited to, transfection,retroviral infection, electroporation, lipofection, bacterial transferand spheroplast fusion. Nucleic acid molecules transformed into cellssuitable for use in the present invention can either remain onextra-chromosomal vectors or can be integrated into the cell genome.

[0046] Expression of a nucleic acid molecule of the present invention ina cell can be accomplished using techniques known to those skilled inthe art. Briefly, the nucleic acid molecule is inserted into anexpression vector in such a manner that the nucleic acid molecule isoperatively joined to a transcription control sequence in order to becapable of effecting either constitutive or regulated expression of thegene when the gene is transformed into a host cell. Construction ofdesired expression vectors can be performed by methods known to thoseskilled in the art and expression can be in eukaryotic or prokaryoticsystems. An expression system can be constructed from control elements,including transcription control sequences, translation controlsequences, origins of replication, and other regulatory sequences thatare compatible with a host cell, operatively linked to nucleic acidsequences using methods known to those of skill in the art. (see, forexample, Sambrook et al., ibid.).

[0047] One aspect of the present invention includes a cell-based assayto identify compounds, referred to herein as “putative regulatorycompounds”, which are capable of regulating actin polymerization in a Tcell. As used herein, the term “putative” refers to compounds having anunknown or previously unappreciated regulatory activity in a particularprocess. As such, the term “identify” is intended to include theparticular selection of any compound, the usefulness of which as aregulatory compound of actin polymerization is determined by a method ofthe present invention.

[0048] One embodiment of the present invention relates to a method toidentify a putative regulatory compound that regulates actinpolymerization in a T cell, comprising: (1) contacting a putativeregulatory compound with a T cell having a T cell receptor chainincluding a ζ chain and an ε chain, to form a “contacted cell”; (2)combining the contacted cell with a molecule capable of inducing thephosphorylation of the ζ chain or the ε chain; and (3) assessing theability of the putative regulatory compound to regulate actinpolymerization in the cell.

[0049] Suitable cells for use with the present invention includemammalian, invertebrate, plant, insect, fungal, yeast and bacterialcells. Preferably cells for use with the present invention includemammalian cells, more preferably human, non-human primate, mouse, rat,sheep and pig cells, and even more preferably mouse and human cells.More preferred cells include T lymphocytes, with murine and human T celllines, murine and human T cell clones, with murine and human T cellhybridomas being even more preferred. In a preferred embodiment,putative regulatory compounds are identified using Jurkat, HPB, cellsexpressing CD25 conjugated to a full length ζ chain (e.g., MM-16.11,MM-16.12, MM-16.2 and MM-16.5), cells expressing CD25 conjugated to a ζchain that lacks the first tyrosine residue in each ITAM (e.g., MM-17.1,MM-17.12, MM-17.2 and MM-17.4), cells expressing CD25-ζY153F (e.g.,MM-18.2, MM-18.4, MM-18.6 and MM-18.7), cells expressing CD25 conjugatedto a full length ε chain (e.g., 17.1 and MM-17.2), cells expressing CD8conjugated to a full length ζ chain (e.g., 122.26), cells expressing CD8conjugated to a ζ chain that lacks residues 95-163 (e.g., T91.10), cellsexpressing CD8 conjugated to a ζ chain that lacks residues 66-100 and129-161 (e.g., B21), cells expressing CD8 conjugated to a ζ chain thatlacks residues 67-126 (e.g., 5.6.4), cells expressing CD8-ζY153E (e.g.,910.7), cells expressing CD8-ζY153F (e.g., 78.12), CD25-ζ, CD8-ζ andCD8-ε chimeric molecules described in Wegener et al. (ibid.), HL-60,H-9, peripheral T cells, PBMC cells, lymph node T cells, splenic Tcells, thymocytes, intraepithelial lymphocytes (IEL) and tumorinfiltrating lymphocytes (TIL).

[0050] Alternatively, cells for use with the present invention caninclude spontaneously occurring variants of normal cells, or geneticallyengineered cells, that have altered signal transduction activity, suchas enhanced responses to particular ligands. Signal transductionvariants of normal cells can be identified using methods known to thosein the art. For example, variants can be selected using fluorescenceactivated cell sorting (FACS) based on the level of calcium mobilizationby a cell in response to a ligand. Genetically engineered cells caninclude recombinant cells of the present invention (described in detailbelow) that have been transformed with, for example, a recombinantmolecule encoding a signal transduction molecule of the presentinvention.

[0051] In certain embodiments, a cell of the present invention istransformed with at least one heterologous nucleic acid molecule.Preferred nucleic acid molecules with which to transform a cell include,but are not limited to, a nucleic acid molecule encoding a chimericprotein comprising a portion of a CD25 molecule peptide bonded to aportion of a ζ chain (CD25-ζ), a portion of a CD8 molecule peptidebonded to a portion of a ζ chain (CD8-ζ), a portion of a CD25 moleculepeptide bonded to a portion of a ε chain (CD25-ε) and a portion of a CD8molecule peptide bonded to a portion of a ε chain (CD8-ε). Preferredcell lines of the present invention include Jurkat or BW-51-47 cellstransfected with nucleic acid molecules encoding such CD25-ζ or CD8-ζchain, and CD25-ε or CD8-ε chimeric molecules.

[0052] In another embodiment, a cell suitable for use in the presentinvention has one or more intracellular signal transduction moleculescapable of transmitting a signal through the cytoplasm of the cell,resulting in actin polymerization. An intracellular signal transductionmolecule as described herein can be produced in a cell by expression ofa naturally occurring gene and/or by expression of a heterologousnucleic acid molecule transformed into the cell.

[0053] A preferred cell of the present invention has, amongst othersignal transduction molecules, tyrosine kinases and adaptor molecules.Suitable tyrosine kinases of the present invention include a tyrosinekinase capable of regulating the activity of a ζ or ε chain, such asenhancing or limiting the ability of a ζ chain to induce calcium releasein a cell. Calcium release or mobilization refers to measurableincreases in intracellular calcium in a cell. Calcium release can bemeasured using methods known to those skilled in the art and generallydescribed in Finkel et al. (Nature 330:6144-6146, 1987). Preferredtyrosine kinases of the present invention include src-family tyrosinekinases and syk-family tyrosine kinases. More preferred tyrosine kinasesinclude Zap-70, Syk, Fyn, Lck, Yes, Hck, fak-B and PI-3 kinase. As usedherein, adaptor molecules enable two other proteins to form a complex(e.g., a three molecule complex). Preferred adaptor molecules of thepresent invention includes Shc, IRS-1, Nck and GRB-2, with Shc beingmore preferred. A preferred cell of the present invention also includesother signalling molecules, preferably 14-3-3 and CSK.

[0054] A preferred cell of the present invention further comprises actinbinding proteins including, but not limited to, focal adhesion kinase,paxillin, tensin, annexin, ezrin, clathrin-H chain, vinculin, talin,zixin, profilin, fractinin, cortactin, AFAP-110, p120, β catenin,connexin43 and cadherins.

[0055] Putative compounds as referred to herein include, for example,compounds that are products of rational drug design, natural productsand compounds having partially defined signal transduction regulatoryproperties. A putative compound can be a protein-based compound, acarbohydrate-based compound, a lipid-based compound, a nucleicacid-based compound, a natural organic compound, a synthetically derivedorganic compound, an anti-idiotypic antibody and/or catalytic antibody,or fragments thereof. A putative regulatory compound can be obtained,for example, from libraries of natural or synthetic compounds, inparticular from chemical or combinatorial libraries (i.e., libraries ofcompounds that differ in sequence or size but that have the samebuilding blocks; see for example, U.S. Pat. Nos. 5,010,175 and 5,266,684of Rutter and Santi, which are incorporated herein by reference in theirentirety) or by rational drug design.

[0056] In a rational drug design procedure, the three-dimensionalstructure of a compound, such as a signal transduction molecule can beanalyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. This three-dimensional structure can then be used topredict structures of potential compounds, such as putative regulatorycompounds by, for example, computer modelling. The predicted compoundstructure can then be produced by, for example, chemical synthesis,recombinant DNA technology, or by isolating a mimetope from a naturalsource (e.g., plants, animals, bacteria and fungi). Potential regulatorycompounds can also be identified using SELEX technology as described in,for example, PCT Publication Nos. WO 91/19813; WO 92/02536 and WO93/03172 (which are incorporated herein by reference in their entirety).

[0057] In particular, a naturally-occurring intracellular signaltransduction molecule can be modified based on an analysis of itsstructure and function to form a suitable regulatory compound. Forexample, a compound capable of regulating the activity of an ITAM cancomprise a compound having similar structure to an ITAM, a tyrosinekinase, an adaptor molecule, 14-3-3 or CSK. Additionally, a compoundcapable of interfering with the association of an ITAM with a targetmolecule can comprise a compound having similar structure to an ITAM, aSH2 domain of a tyrosine kinase, an adaptor molecule, 14-3-3 or CSK.

[0058] The conditions under which a cell of the present invention iscontacted with a putative regulatory compound, such as by mixing, areconditions in which the cell can form actin polymers if essentially noother regulatory compounds are present that would interfere with actinpolymerization. Achieving such conditions is within the skill in theart, and includes an effective medium in which the cell can be culturedsuch that the cell can exhibit cytoskeletal rearrangement. For example,for a mammalian cell, effective media are typically aqueous mediacomprising Dulbecco's modified Eagle's medium containing 10% fetal calfserum.

[0059] Cells of the present invention can be cultured in a variety ofcontainers including, but not limited to, tissue culture flasks, testtubes, microtiter dishes, and petri plates. Culturing is carried out ata temperature, pH and carbon dioxide content appropriate for the cell.Such culturing conditions are also within the skill in the art. Forexample, for Jurkat cells, culturing can be carried out at 37° C., in a5% CO₂ environment.

[0060] Acceptable protocols to contact a cell with a putative regulatorycompound in an effective manner include the method of contact, thenumber of cells per container contacted, the concentration of putativeregulatory compound(s) administered to a cell, the incubation time ofthe putative regulatory compound with a cell, the concentration ofstimulatory molecules administered to a cell, and the incubation time ofthe stimulatory molecules with a cell. Determination of such protocolscan be accomplished by those skilled in the art abased on variables suchas the size of the container, the volume of liquid in the container, thetype of cell being tested and the chemical composition of the putativeregulatory compound (i.e., size, charge etc.) being tested.

[0061] Preferred methods for contacting a cell include electroporation,microinjection, cellular expression (i.e., using an expression systemincluding naked nucleic acid molecules, recombinant virus, retrovirusexpression vectors and adenovirus expression), use of ion pairing agentsand use of detergents for cell permeabilization.

[0062] A suitable number of cells to be used with the present methodincludes a number of cells that enables one to detect a change incytoskeletal structure using a detection method of the present invention(described in detail below). A more preferred number of cells includesbetween about 1 and 1×10⁶ cells per well of a 96-well tissue culturedish. Following addition of the cells to the tissue culture dish, thecells can be pre-incubated at 37° C., 5% CO₂ for between about 0 toabout 72 hours.

[0063] A suitable amount of putative regulatory compound(s) that issufficient to regulate the activity of a signal transduction moleculeinside the cell such that the regulation is detectable using a detectionmethod of the present invention is electroporated into the cells usingmethods standard in the art based on the type of putative regulatorycompound and the type of recipient cell. A preferred amount of putativeregulatory compound(s) comprises between about 1 nM to about 10 mM ofputative regulatory compound(s) per well of a 96-well plate.

[0064] In another embodiment of the method of the present invention,cells suitable for use in the present invention are stimulated withstimulatory molecules capable of binding to cell surface receptors ofthe present invention to initiate a signal transduction pathway andcreate a cellular response. Preferably, cells are stimulated with astimulatory molecule following contact of a putative regulatory compoundwith a cell. Suitable stimulatory molecules can include, for example,hormones, growth factors, antigens, peptides, ions, otherdifferentiation agents and other cell-type specific mitogens. Preferredstimulatory molecules include, but are not limited to, an antibodyspecific for a T cell receptor, an antibody specific for a ζ chain, anantibody specific for an ε chain, mitogens, lectins and majorhistocompatibility molecules associated with an antigenic peptide, andmixtures thereof. Particularly preferred stimulatory molecules of thepresent invention include, an antibody specific for a T cell receptor,an antibody specific for a ζ chain, an antibody specific for an ε chain,and mixtures thereof. A suitable amount of stimulatory molecule to addto a cell depends upon factors such as the type of ligand used (e.g.,monomeric or multimeric; permeability, etc.) and the receptor beingtargeted (e.g., abundance of the receptor on a cell and the number ofligand binding sites/receptor). Preferably, between about 1 microgram(μg) and about 5 μg of ligand is added to about 1×10⁶ cells.

[0065] The cells are allowed to incubate under standard conditions(based upon, for example, time, pH, etc.) for a suitable length of timeto allow the stimulatory molecule to stimulate a signal transductionpathway. A preferred incubation time is between about 30 seconds toabout 24 hours.

[0066] The method of the present invention includes determining if aputative regulatory compound is capable of regulating actinpolymerization. Such methods include: actin polymerization analysis;cellular analysis; and performing protein phosphorylation or activationassays.

[0067] In one embodiment, the method of the present invention comprisesdetecting actin polymerization in a cell by determining alteration in acell contacted with a putative regulatory compound, compared with a cellthat has not been contacted with the same compound. For example, tomeasure the effectiveness of a putative regulatory compound forcontrolling actin polymerization, one can observe the adhesiveness,growth, shape and motility characteristics of contacted and uncontactedcells. The extent of adhesiveness of a cell can be determined by whethera cell can be dislodged from a substratum by shaking or knocking theculture surface, or whether enzyme treatment, such as trypsin, isrequired to dislodge a cell from a substratum. Methods to dislodge cellsfrom culture surfaces are well known in the art. Changes in cell growthcan be determined by: counting the number of live cells after a certainperiod of time (by, for example, trypan blue staining or hoerschtstaining); by determining the extent of tritiated thymidine uptake by acell; by determining the amount of cytokine secretion by a cell (e.g.,measuring IL-2 secretion); and/or by measuring the size of a cell aftera certain period of time. Changes in cell shape can be determined byviewing cells to assess the flattening of cells on a substratum and/orthe formation of cellular extensions, such as pseudopodium, filopodiumand lamellipodium. Changes in cell motility can be determined by viewingthe direction and distance a cell has traveled on a substratum during acertain period of time. Typically, such movement is associated with theformation of cellular extensions.

[0068] In another embodiment, the method of the present inventioncomprises detecting actin polymerization in a cell by determiningchanges in actin polymerization and/or organization. In such a method,the extent of actin polymerization and/or the organization of actinfilaments are compared in cells contacted with a putative regulatorycompound and cells not contacted with such a compound. The actinfilaments are visualized by contacting the cells with labelledphalloidin (e.g., rhodamine conjugated phalloidin), which bindsspecifically to F-actin, using methods described herein (see, Example 1below). Alternatively, actin filaments can be visualized using labelledantibodies that specifically bind to actin monomers or polymers, usingmethods known to those in the art. Additional methods includevisualizing actin polymerization by electron microscopy, falling ballviscometry, spectrophotometry and sedimentation.

[0069] In yet another embodiment, the method of the present inventioncomprises detecting actin polymerization in a cell by determiningassociation with and modulation of signal transduction proteinsincluding, but not limited to, focal adhesion kinase, paxillin, tensin,annexin, ezrin, clathrin-H chain, vinculin, talin, zixin, profilin,fractinin, cortactin, AFAP-110, p120, β catenin, connexin43, cadherins,PI-3Kα, TCR, Syk, Zap-70, Fyn, Shc, IRS-1, Nck, GRB-2, Lck, VAV, GAP,Raf, Ras, MEK, MEKK, MAPK, p38, JNKK, JNK, jun-B, PLA2, JAK1, JAK2,JAK3, Tyk1, Tyk2, STATs, Myc, Jun, Ets-1, Elk-1, CREB, ATF-2, Yes, Hck,Src, CaM Kinase II, S6-K, sphingomyelinase, casein kinase, PKC, PI-3Kγ,SOS, CD45, HCP, Ssp, Syp, PLCγ1, PLCγ2, PLCβ1, PLCβ2, PLCβ3, PLCβ4,PLA2, Grb2, C5aR, IL-8R, MIP1αR, MIP1βR, MCP-1R, MCP-3R, PAFR, FMLPR,LTB₄R, GRPR, Fas receptor, Fas ligand, NFκ-B, SHP-76, N-FAT, AP-1, CD7,CD5, tumor necrosis factor receptor, CD40 ligand, CD28, CD2, integrinsand addressins. As used herein, modulation of a signal transductionprotein refers to, for example, the phosphorylation of a molecule or theassociation of a molecule to another signalling molecule. One method todetermine modulation of a signal transduction molecule is to determinethe phosphorylation state of the molecule using methods and reagentsknown to those of skill in the art. For example, phosphorylation can bedetected using antibodies specific for phosphorylated amino acidresidues. Alternatively, polymerized actin can be isolated bysedimentation and the actin-associated proteins can be identified.

[0070] Alternatively, the method of the present invention includesdetermining if a putative regulatory compound is capable of regulating Tcell function. Such methods include determining IL-2 production by a Tcell; and assessing T cell apoptosis, growth, adhesion, differentiation,proliferation and/or homing.

[0071] The method of the present invention is particularly useful forregulating actin polymerization in cells involved in diseases,including, but not limited to, tumorigenesis, immunoproliferativediseases, immunodeficiency diseases, cancers, autoimmune diseases,infectious diseases, allergic responses and graft rejection. Inparticular, the present method protects an animal from diseasesincluding, for example, rheumatoid arthritis, SLE, vasculitis,scleroderma, solid tumors, hematopoietic malignancies, acute and chronicgraft rejection, AIDS, asthma and allergic rhinitis.

[0072] Another aspect of the present invention includes a kit toidentify compounds capable of regulating actin polymerization, in acell, such actin polymerization involving in some respect, ζ and/or εchains of a TCR. Such a kit includes: (a) a cell comprising a T cellreceptor chain selected from the group consisting of a ζ chain, an εchain, and actin monomers; and (b) a means for detecting thepolymerization of such actin monomers. Such a means for detecting actinpolymerization are described in detail herein and are known to those ofskill in the art. Suitable cells for use with a kit of the presentinvention include cells described in detail herein. A preferred cell foruse with a kit includes, Jurkat, HPB, MM-16.11, MM-16.12, MM-16.2,MM-16.5, 17.1, MM-17.2, 122.26, HL-60, H-9, peripheral T cells, PBMCcells, lymph node T cells, splenic T cells, thymocytes, intraepitheliallymphocytes (IEL) and tumor infiltrating lymphocytes (TIL).

[0073] The present invention also includes the determination as towhether a putative regulatory compound is capable of regulating abiological response in a mammal. Such a method entails administering aputative regulatory compound to an animal, such compound being shown,using an assay of the present invention, to regulate actinpolymerization in a cell. Such a determination is useful for determiningconditions under which a putative regulatory compound can beadministered to an animal as a formulation of the present invention. Inparticular, a putative regulatory compound can be administered to ananimal to determine if the compound is capable of regulating, forexample, an immune response, an allergic response and/or graft rejectionin the animal. Acceptable protocols to administer putative regulatorycompounds to test the effectiveness of the compound include individualdose size, number of doses, frequency of dose administration, and modeof administration. Determination of such protocols can be accomplishedby those skilled in the art. A suitable single dose is a dose that iscapable of altering a biological response in an animal when administeredone or more times over a suitable time period (e.g., from minutes todays or weeks). Preferably, a dose comprises from about 1 nanogram ofthe compound per kilogram of body weight (ng/kg) to about 1 gram ofcompound per kilogram of body weight (gm/kg), more preferably 100 ng/kgto about 100 milligrams/kilogram (mg/kg), and even more preferably fromabout 10 micrograms of compound per kilogram of body weight to about 10mg/kg. Modes of administration can include, but are not limited to,intraarticular, intraperitoneal, subcutaneous, rectally, intradermal,intravenous, nasal, oral, transdermal and intramuscular routes. Aputative regulatory compound can be combined with other components suchas a pharmaceutically acceptable carrier as described in detail herein,prior to administration to an animal.

[0074] In another aspect of the present invention, the present inventionincludes conducting a toxicity test on an animal to determine thetoxicity of a putative regulatory compound. Toxicity tests for putativeregulatory compounds can be performed, for example, on animals after aputative regulatory compound has been determined to have an effect atthe cellular level on signal transduction, such as the regulation ofcellular inflammatory responses. Such toxicity tests are within theskill of the art, and generally involve testing the toxicity of acompound in vivo or in vitro. A suitable method for testing the toxicityof a putative regulatory compound in vivo can involve scientificallycontrolled administration of the putative regulatory compound to anumber of animals and a period of observance in which the effects of thecompound on various aspects of the animal's biological functions (e.g.,occurrence of tissue damage, functioning of organs and death) are noted.Suitable methods for testing the toxicity of a putative regulatorycompound in vitro can involve scientifically controlled administrationof the putative regulatory compound to a cell and subsequent measurementof cell function, cytotoxicity, or cell death. Cell function can bemeasured by any one of a wide range of assays which will be apparent toone of skill in the art, several of which are herein disclosed (e.g.,tyrosine phosphorylation, calcium mobilization, proliferation andcytokine secretion assays). Methods to measure cytotoxicity are wellknown in the art and include measurement of the ability to reducechromogenic substrates such as the tetrazolium-based MTT orsulphorhodamine blue, ATP-bioluminescence assays and fluorescenceassays, for example using the Fluorescent Green Protein, among manyother readily available assays (see, for example, Bellamy, Drugs44(5):690-708, 1992, which is incorporated herein by reference in itsentirety). Methods to measure cell death include, for example, coomassieblue staining, acridine orange staining, terminal deoxynucelotidyltransferase (TDT) assays for measuring DNA fragmentation, neutral redexclusion, and measuring changes in forward light scatter in a flowcytometer.

[0075] Another aspect of the present invention comprises administeringto an animal a formulation capable of regulating actin polymerization. Aformulation of the present invention is particularly useful forpreventing or treating diseases involving abnormal T cell activity,growth or migration.

[0076] An effective administration protocol (i.e., administering aformulation in an effective manner) comprises suitable dose parametersand modes of administration that result in prevention or treatment of adisease. Effective dose parameters and modes of administration can bedetermined using methods standard in the art for a particular disease.Such methods include, for example, determination of survival rates, sideeffects (i.e., toxicity) and progression or regression of disease. Forexample, the effectiveness of dose parameters and modes ofadministration of a formulation of the present invention can bedetermined by assessing response rates. Such response rates refer to thepercentage of treated patients in a population of patients that respondwith either partial or complete remission.

[0077] In accordance with the present invention, a suitable single dosesize is a dose that is capable of preventing or treating an animal witha disease when administered one or more times over a suitable timeperiod. Doses can vary depending upon the disease being treated. Forexample, in the treatment of cancer, a suitable single dose can bedependent upon whether the cancer being treated is a primary tumor or ametastatic form of cancer.

[0078] It will be obvious to one of skill in the art that the number ofdoses administered to an animal is dependent upon the extent of thedisease and the response of an individual patient to the treatment. Forexample, in the case of cancer, a large tumor may require more dosesthan a smaller tumor. In some cases, however, a patient having a largetumor may require fewer doses than a patient with a smaller tumor, ifthe patient with the large tumor responds more favorably to thetherapeutic composition than the patient with the smaller tumor. Thus,it is within the scope of the present invention that a suitable numberof doses, as well as the time periods between administration, includesany number required to cause regression of a disease.

[0079] Formulations can be administered directly to a cell in vivo or exvivo or systemically. Preferred methods of systemic administration,include intraarticular, intravenous injection, aerosol, oral andpercutaneous (topical) delivery. Intravenous injections can be performedusing methods standard in the art. Aerosol delivery can also beperformed using methods standard in the art (see, for example, Striblinget al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which isincorporated herein by reference in its entirety). Oral delivery can beperformed by complexing a therapeutic composition of the presentinvention to a carrier capable of withstanding degradation by digestiveenzymes in the gut of an animal. Examples of such carriers, includeplastic capsules or tablets, such as those known in the art. Topicaldelivery can be performed by mixing a formulation of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

[0080] The following examples are provided for the purposes ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1

[0081] This example describes the association of TCR-ζ chain with thedetergent insoluble cell fraction upon T cell receptor ligation.

[0082] A. Cell Activation and Lysis

[0083] Freshly isolated thymocytes or lymph node T cells from normaladult C57B1/6 mice were washed 3 times in BSS+5% fetal calf serum (FCS)and incubated in the presence or absence of an anti-pan-TCR-β (H57-597;hereafter referred to as anti-αβTCR antibody; Kubo et al., J. Immunol.142:2736-2742, 1989) antibody at 5 μg/10⁶ cells, and/or cross-linkinggoat anti-mouse antibody (obtained from Sigma Chemical Co., St. Louis,Mo.) at 20 μg/ml, for 30 min at 4° C., with at least two washes withBSS+5% FCS after each antibody incubation. The cells were then incubatedfor 15 min at 37° C., solubilized with 0.5% NP-40 in a Tris-bufferedsaline solution (TBS; 150 mM NaCl, 10 mM Tris, pH 7.3) containingprotease and phosphatase inhibitors (0.2 mM VO₃, 10 mM NaF, 10 mMtetrasodium pyrophosphate, 1 mM PMSF, and 1 μg/ml each of Aprotinin,Leupeptin, and α-1-antitrypsin) and centrifuged at 10,000 rpm for 10 minto pellet the insoluble material.

[0084] Prior to separation by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the insoluble cellpellet was resolubilized by boiling in non-reducing sample buffer.Immunoprecipitation of the detergent soluble fraction was performed withSepharose-conjugated anti-TCR-ζ mAb (H146-968; Rozdzial et al., J.Immunol. 153:1563-1580, 1994).

[0085] B. Gel Electrophoresis and Immunoblotting

[0086] The proteins contained in the isolated pellet and the product ofthe anti-TCR-ζ immunoprecipitation performed in step A were boiled innon-reducing sample buffer and, at 1-5×10⁷ cell equivalents/lane, wereseparated under non-reducing conditions by 10% SDS-PAGE gelelectrophoresis. Electrophoretic transfer of protein to 0.2 μmnitrocellulose filters was performed in 48 mM Tris, 39 mM glycine, 1.3mM SDS, and 20% methanol, at room temperature under constant current(150-200 mA) for 2 hrs. Efficiency of transfer was monitored by transferof prestained markers, by reversible staining of the blots withPonceau-S, and by silver staining of the electroeluted gels. The filterswere then quenched in blotting buffer composed of 125 mM NaCl and 25 mMTris, pH 7.6 (TS), and 5% skim milk, or with 5% crystallized bovineserum albumin (BSA). Following electrotransfer and quenching, thenitrocellulose filters were immunoblotted with specific antibodies (1μg/ml) to TCR-ζ for 3 hr, and washed in TS-0.05% Tween-20. The washedfilters were incubated with ¹²⁵I-protein A (4×10⁵ cpm/ml) in quenchingbuffer for 1 hr, and washed as above. The blots were then dried andexposed to Kodak XAR-2 film at −70° C.

[0087] C. Densitometry

[0088] Densitometric analysis (corrected for loading of unequal cellequivalents) was performed on the resulting immunoblots using aMacIntosh image scanner. The scans were than interfaced with a MacIntoshcomputer and densitometric analysis carried out using the NIH Image 1.49program (NIH, Bethesda, Md.) for one-dimensional scanning.

[0089] D. Results

[0090] The results of the immunoblot analysis indicate that about 7% ofTCR-ζ protein sedimented with the detergent insoluble pellet fromresting thymocytes and lymph node T cells. Upon activation of thymocytesand lymph node T cells, an average of about 16% and about 43% of TCR-ζprotein, respectively, associated with the detergent insoluble pelletand was depleted from the supernatant relative to resting cells. Theactivation-induced association with the detergent insoluble pelletoccurred with a concomitant depletion of TCR-ζ protein from thedetergent soluble fraction. Treatment of thymocytes or lymph node Tcells with the anti-TCR-ζ antibody or GAM alone did not substantiallyincrease the amount of TCR-ζ in the pellet relative to untreatedcontrols.

[0091] Thus, upon ligation of TCR, the amount of TCR-ζ in the detergentinsoluble pellet increased an average of two-fold (16%) and six-fold(43%) compared with non-ligated samples. In comparison, in resting Tcells, TCR-ζ protein was predominantly localized to the detergentsoluble material. In addition, the amount of TCR-ζ in the insolublepellet from activated lymph node T cells is significantly greater thanthat isolated from activated thymocytes.

Example 2

[0092] This example describes the association of CD8-ε chimeric proteinswith the cytoskeleton upon TCR ligation.

[0093] A T cell hybridoma that expresses a CD8-ε chimeric moleculecomprising a full-length ε chain was incubated in the presence orabsence of an anti-CD8 antibody (53.6.72). Each sample was thenincubated in the presence of a goat anti-rat antibody (GAR; obtainedfrom Jackson Immuno Research Labs, West Grove, PN) to cross-linkanti-CD8 antibody bound to the surface of the hybridomas. The cells werethen lysed and immunoblots prepared as described in Example 1.Immunoblots were performed using anti-CD8 antibody.

[0094] The results indicate that a CD3-ε chain can associate with theinsoluble pellet in response to TCR ligation. Thus, the ability toassociate with the cytoskeleton is shared between different chains ofthe TCR.

Example 3

[0095] This example describes the association of TCR-ε with cytoskeletonindependent of other chains of the TCR.

[0096] Chimeric constructions were assembled by polymerase chainreaction (PCR) as described (Beaufils et al. EMBO J. 12:5105-5112,1993). Sequences of the various PCR products were confirmed using thedideoxy-chain termination method. A first chimeric DNA construct,referred to herein as pCD25/ζ, comprises a nucleic acid moleculeencoding the extracellular and transmembrane regions of CD25 ligated toa nucleic acid molecule encoding the cytoplasmic domain of TCR-ζ, clonedinto a pSRa-neo expression vector. pCD25/ζ encodes for a proteinreferred to herein as CD25/ζ, which is illustrated in FIG. 1. Referringto FIG. 1, the a, b, and c regions represent the three ITAMs of thecytoplasmic domain of the TCR-ζ chain, each region having two tyrosines(Y).

[0097] A second chimeric DNA construct, referred to herein aspCD25/ζY153F, comprises a nucleic acid molecule identical to pCD25/ζexcept the tyrosine at residue 153 was substituted for a phenylalanine(illustrated in FIGS. 1 and 2). pCD25/ζY153F encodes for a proteinreferred to herein as CD25/ζY153F.

[0098] pCD25/ζ and pCD25/ζY153F were transfected into the BW 5147α⁻β⁻thymoma (described in White et al., J. Immunol. 143:1822-1825, 1989)and selected in the presence of G418-sulfate using the methods describedin Wegener et al. (Cell 68:83-95, 1992).

[0099] The T cell hybridomas expressing CD25/ζ and CD25/ζY153F wereincubated in the presence or absence of anti-CD25 antibody (obtainedfrom AMAC, Inc., Westbrook, Me.). The cells were then incubated in thepresence of GAR antibody to cross-link antibody-bound surface CD25. Thesamples were then lysed and immunoblotted using the methods described inExample 1. Immunoblots were performed using anti-TCR-ζ antibody oranti-phosphotyrosine antibody (obtained from Sigma Chemical Co., St.Louis, Mo.).

[0100] The results indicate that CD25/ζ associated with the detergentinsoluble pellet in response to ligation of the extracellular domain ofCD25. The results further indicate that CD25/ζY153F could not associatewith the cytoskeleton. Thus, the results indicate that other componentsof the TCR are not required for the interaction of ζ with thecytoskeleton. Moreover, the results indicate that the tyrosine atresidue 153 of the ζ chain is important for the association of the ζchain to the cytoskeleton.

Example 4

[0101] This example describes the cytoskeletal component involved inTCR-ζ binding to the cytoskeleton upon TCR ligation.

[0102] Cleared lysates of resting and activated T cells were preparedusing the method described in Example 1. TCR-ζ protein was precipitatedfrom the detergent soluble fraction using the anti-TCR-ζ antibodydescribed above. The resulting precipitate was resolved by SDS-PAGE gelelectrophoresis and immunoblotted with either anti-actin antibody(kindly provided by Dr. B. Jockusch, Braunschweig, Germany) oranti-tubulin antibody (obtained from Sigma Chemical Co., St. Louis, Mo.)or anti-TCR-ζ antibody.

[0103] Based upon the immunoblot results, actin was shown toco-precipitate with TCR-ζ, but not tubulin. The association of actinwith TCR-ζ increased significantly in response to TCR ligation, underconditions favoring cytoskeleton depolymerization (i.e., incubation inthe presence of cytoskeletal poisons). In the absence of cytoskeletalpoisons, binding of TCR-ζ protein to actin was not increased despite TCRligation. These results indicate that activation induces a change, inTCR-ζ, actin or an intermediary molecule, that promotes the interactionbetween TCR-ζ and the cytoskeleton. The results also indicate that theactin which co-precipitated with TCR-ζ after TCR ligation separated at aslightly higher molecular weight than actin isolated from non-TCRligated cells, thereby indicating that the actin has undergoneposttranslational modification upon cell activation.

[0104] Together, these results demonstrate an association of TCR-ζ withmicrofilaments and regulation of this association by T cell activation.These data also indicate that actin monomers, dimers or short filamentscontain a binding site for TCR-ζ interaction.

Example 5

[0105] This example describes the molecular mechanisms and structuralinteractions that mediate the association of TCR-ζ with the detergentinsoluble pellet.

[0106] Lymph node T cells or hybridoma cells expressing pCD25/ζdescribed in Example 3 were incubated in 5 μg/ml cytochalasin D andnocodazole for 1.5 hrs before or after activation with, respectively, ananti-αβTCR antibody or anti-CD25 antibody and cross-linking GAM or GARantibody. The cells were then lysed and both the detergent soluble andinsoluble fractions were immunoblotted according to the methodsdescribed in Example 1. Immunoblots were performed using anti-actinantibody to detect the co-precipitation of actin with TCRζ or anti-TCR-ζantibody.

[0107] A. Detergent Insoluble Fractions

[0108] Association of TCR-ζ with the detergent insoluble pellet wasunaffected in resting and activated cells treated with cytochalasin Dand nocodazole after activation, relative to untreated controls cellsamples. TCR-ζ, however, did not associate with the detergent insolublepellet in cells treated with cytochalasin D and nocodazol prior toactivation. The data indicate a direct or indirect involvement of thecytoskeleton in the association between TCR-ζ and the detergentinsoluble fraction. Cells treated with the cytoskeleton poisonsconsistently showed lower TCR-ζ-cytoskeleton association thanunactivated controls.

[0109] B. Detergent Soluble Fractions

[0110] The results indicate that TCR-ζ remaining in the detergentsoluble supernatant of samples co-immunoprecipitates with actin eitherin the presence or absence of cytochalasin D and nocodazol, underresting or activating conditions. The amount of soluble actinco-precipitating with TCR-ζ, under conditions of cytoskeletaldepolymerization, increased significantly in cell lysates from activatedrelative to resting thymocytes.

[0111] Thus, the results indicate that treatment of the cells withcytoskeletal poisons disrupted association of TCR-ζ with the pellet incells treated before, but not after TCR ligation. Partial disruption ofthe association of TCR-ζ with the pellet was also seen in thymocytes andperipheral lymph node T cells treated with cytoskeletal poisons,indicating involvement of the cytoskeleton, specifically microfilaments,in the interaction with TCR-ζ.

Example 6

[0112] This example describes the requirement for the third ITAM ofTCR-ζ in the association of TCR-ζ with cytoskeleton.

[0113] BW 5147 α⁻β⁻thymoma cells transfected with truncated TCR-ζ(ζD66-114 deleted in residues 66-114; or ζD66-157, deleted in residues66-157; described in Wegener et al., ibid.; illustrated in FIG. 2) wereincubated in the absence or presence of anti-αβTCR antibody and GAM, orGAM alone. The cells were then lysed and immunoblotted with anti-TCR-ζantibody using the methods described in Example 1. The foregoing methodwas repeated in about 3 different experiments.

[0114] The results indicate that only the full length TCR-ζ and thetruncated TCR-ζ encoded by the ζD66-114 construct (not expressing thefirst ITAM) associated with the cytoskeleton upon activation. The TCR-ζencoded by the ζD66-157 construct did not associate with thecytoskeleton in response to TCR ligation. These results indicate thatthe region of TCR-ζ containing the ITAMs, rather than the membraneproximal or distal regions of the polypeptide, is required forcytoskeletal association.

[0115] Experiments were performed to confirm the requirement of thethird ITAM for TCR-ζ association with cytoskeleton. BW 5147 α⁻β⁻thymomacells transfected with a nucleic acid molecule encoding a truncatedCD8/ζ chimera (CD8/ζD67-126, expressing only intact ζc domain; describedin Wegener et al., ibid.; illustrated in FIG. 2) were stimulated, lysedand immunoblotted using the methods described immediately above. Theresults indicated that the CD8/ζ chimera containing only the third ITAMof the ζ chain associated with the cytoskeleton, thereby indicating thatthe third activation motif of TCR-ζ is sufficient for cytoskeletalassociation.

Example 7

[0116] This example describes the requirement for phosphorylation of thedistal tyrosine of the third ITAM of TCR-ζ in cytoskeleton association.

[0117] Tyrosine phosphorylated-proteins were detected in freshlyisolated thymocytes or lymph node T cells from normal adult C57B1/6 miceand in CD25/ζ-expressing T cell hybridomas, incubated in the absence orpresence of anti-αβTCR or anti-CD8-ε (145-2C11; Leo et al., Proc. Natl.Acad. Sci. USA 84:1374-1378, 1987; illustrated in FIGS. 1 and 2)antibodies and/or GAM, and lysed, as described in Example 1.Immunoprecipitation of the detergent soluble fraction was thenperformed, in series, with agarose-linked anti-phosphotyrosine antibody(Ab-1, Oncogene Science, Uniondale, N.Y.) and Sepharose-conjugatedanti-TCR-ζ antibody (H146-968). Following electrotransfer, thenitrocellulose filters were immunoblotted with specific antibodies toTCR-ζ or phosphotyrosine (Ab-2, Oncogene Science, Uniondale, N.Y.).

[0118] As indicated in FIG. 1, each ITAM TCR-ζ of has two tyrosines.Analysis of the detergent insoluble pellet revealed an increase intyrosine phosphorylated TCR-ζ in the pellets of activated compared toresting thymocytes and lymph node T cells. In addition, analysis of theCD25/ζ chimera after receptor ligation revealed an association oftyrosine-phosphorylated TCR-ζ with the cytoskeleton. The resultsindicate that inductive tyrosine phosphorylation precedes cytoskeletalassociation or, alternatively, occurs as a result of this association.Finally, results obtained using the CD8/ζY153F (described in Wegener etal., ibid. or CD25/ζY153F chimeras, which have substitutions of thedistal, COOH-terminal tyrosine, indicated that the removal of the distaltyrosine almost completely abrogated cytoskeletal association, therebyindicating that tyrosine phosphorylation of the third ITAM plays acritical role in TCR-ζ association with the cytoskeleton.

Example 8

[0119] This example describes the association of tyrosine phosphorylatedTCR-ζ with the actin cytoskeleton in a cell-free system.

[0120] Freshly isolated murine thymocytes or lymph node T cells, orCD25/ζ-expressing T cell hybridomas, were washed 3 times in BSS andincubated in the presence or absence of cytochalasin D for 1.5 hrs at 4°C., solubilized with 0.5% NP-40 in a Tris buffered saline solution (TBS;150 mM NaCl, 10 mM Tris, pH 7.3) containing protease and phosphataseinhibitors (0.2 mM VO₃₁ 10 mM NaF, 1 mM PMSF, and 1 mg/ml each ofAprotinin, Leupeptin, and α-1-antitrypsin) and centrifuged at 10,000 rpmfor 10 min to pellet the insoluble material. The detergent solublefraction was then incubated with or without MgATP (0.5 mM MgSO₄ and 5 mMATP), Mg²⁺ or ATP alone, incubated at 37° C. for 15 min and centrifugedat 10,000 rpm for 10 min to pellet the newly polymerized material. Toincrease actin polymerization, the Mg²⁺ concentration was increased to 2mM. Certain samples were further incubated with 5 M EDTA to chelateexisting Mg²⁺. Immunoprecipitation of the detergent soluble fractionwith anti-TCR-ζ antibody was then performed as described in Example 4.The pellet and the anti-TCR-ζ immunoprecipitate were resolved bySDS-PAGE gel electrophoresis and immunoblotted with anti-TCR-ζ antibody.

[0121] The results indicated that the addition of exogenous MgATPinduced the association of TCR-ζ with the detergent insoluble pellet anda corresponding depletion of TCR-ζ from solution was detected within 1minute of incubation. Optimal association was determined by time courseexperiments to be after 10-15 minutes at 37° C., similar to that seen inintact cells after ligation of the TCR receptor (see Example 1) Almostno binding was observed at MgATP concentrations below 0.1 mM, whereas at1 mM ATP and above, association of TCR-ζ with the lysis pellet appearedoptimal. Results obtained using cell lysates incubated with Mg²⁺ and/orATP in the presence or absence of EDTA, indicated that the addition ofMg²⁺ or nucleotide contributed to TCR-ζ precipitation in vitro. In thepresence of 2 mM Mg²⁺ alone, actin polymerization was induced in vitrowithout additional TCR-ζ binding, indicating that actin polymerizationis not sufficient for TCR-ζ association.

[0122] The CD25/ζ chimera that co-sediments with the actin cytoskeletonin the in vitro activating (MgATP) condition was depleted from thepellet under actin depolymerizing conditions. In addition, themicrofilament association of the CD25/ζ chimera was abrogated by thesubstitution of tyrosine 153 for phenylalanine. These data providefurther evidence that the actin cytoskeleton is specifically involved inassociation with TCR-ζ in response to T cell activation. Furthermore,the results indicate that tyrosine phosphorylation in the third ITAMplays a critical role in this association.

Example 9

[0123] This example describes that the association of TCR-ζ tocytoskeleton is specific to mature T cells.

[0124] Unfractionated thymocytes from normal adult C57B1/6 mice wereincubated for 30 min at 4° C. with a biotin-labeled anti-αβTCR antibody(H57-597), washed three times, labeled with streptavidin R-phycoerythrin(Tago, Inc., Burlingame, Calif.) for 10 min at 4° C., and sorted on aCoulter 751 flow cytometer at 4° C. to separate the immature (TCR^(low))and mature (TCR^(high)) T cell populations. The mature and immaturethymocytes were then analyzed and found to be 96% and 76% homogeneous,respectively.

[0125] Flow cytometric profiles of mouse thymocytes labeled withfluorescent anti-αβTCR (H57-597) antibody, before (---) sorting intosubpopulations bearing low (dark gray) or high (light gray) antigenreceptor levels are shown in FIG. 3. Receptor expression was notmodulated during the course of the cell sort, as shown in thecross-linked, but unsorted, sample (—). Thus, conditions of TCR ligationthat induce TCR-ζ-cytoskeleton association are not accompanied byreceptor modulation from the cell surface.

[0126] Sorted populations were lysed according to the method describedin Example 1, and the detergent insoluble pellets were immunoblottedwith anti-TCR-ζ antibody. Despite analysis of eight times the cellequivalency of the other populations, ligation of the TCR receptor onimmature thymocytes did not induce detectable association of TCR-ζ withthe cytoskeleton. Most of the TCR-ζ from this immature populationremained in solution.

Example 10

[0127] This example describes that the association of TCR-ζ withcytoskeleton is correlated with late events of T cell activation.

[0128] Microcultures of 0.25 ml were prepared containing 10⁵ respondingT cells expressing CD8/ζ, CD8/ζD67-126, CD8/ζY153E, CD25/ζ orCD25/ζY153E. For stimulation, microtiter wells were precoated with 50 μlof a phosphate-buffered saline solution containing varyingconcentrations of purified anti-CD8-α antibody (19/178) or anti-hCD25antibody (B1.49.9) to cross-link the CD8/ζ, CD8/ζD67-126 and CD8/ζY153E,or CD25/ζ and CD25/ζY153E, respectively, on the surface of the T cells.Control wells were coated with an irrelevant antibody. After 2 hours atroom temperature and 1 hr at 4° C., the wells were washed three timeswith fetal calf serum-containing tissue culture medium. Control wellswere coated with an irrelevant antibody. After 24 hours in culture, thesupernatants were harvested and assayed for IL-2 content using methodsgenerally described in Gillis et al. (J. Immunol. 120:2027-2032, 1978).A concentration of 1 U/ml of IL-2 was the minimum detectable in thisassay.

[0129] The results indicate that IL-2 production correlates with TCR-ζassociation with the cytoskeleton. Referring to FIG. 4, at maximalstimulation, cells expressing the deleted (CD8/ζD67-126; closed circles)and substituted (CD8/ζY153E; closed triangles) constructs produced,respectively, 40- and 100-fold less IL-2 than the wild-type CD8/ζ (opencircles and triangles). Similar results were obtained using cellsexpressing CD25/ζY153F when compared with cells expressing wild-typeCD25/ζ (FIG. 5). Interestingly, receptor ligation of the CD25/ζY153Fconstruct induced tyrosine phosphorylation of a set of proteins that wasqualitatively and quantitatively indistinguishable from that induced inthe intact CD25/ζ chimera, indicating that decreased IL-2 production byCD25/ζ Y153F was not due to the inability of CD25/ζY153F to bind toand/or activate critical tyrosine kinases. These data show thatcytoskeletal association by ITAM-containing motifs is correlated withlate events of T cell activation.

1 33 1 26 PRT Mus musculus 1 Glu Arg Arg Arg Gly Lys Gly His Asp Gly LeuTyr Gln Gly Leu Ser 1 5 10 15 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 2025 2 27 PRT Mus musculus 2 Asn Lys Glu Arg Pro Pro Pro Val Pro Asn ProAsp Tyr Glu Pro Ile 1 5 10 15 Arg Lys Gly Gln Arg Asp Leu Tyr Ser GlyLeu 20 25 3 27 PRT Mus musculus 3 Glu Thr Ala Ala Asn Leu Gln Asp ProAsn Gln Leu Tyr Asn Glu Leu 1 5 10 15 Asn Leu Gly Arg Arg Glu Glu TyrAsp Val Leu 20 25 4 28 PRT Mus musculus 4 Lys Gln Gln Arg Arg Arg AsnPro Gln Glu Gly Val Tyr Asn Ala Leu 1 5 10 15 Gln Lys Asp Lys Met AlaGlu Ala Tyr Ser Glu Ile 20 25 5 28 PRT Mus musculus 5 Glu Arg Arg ArgGly Lys Gly His Asp Gly Leu Tyr Asp Ser His Phe 1 5 10 15 Gln Ala ValGln Phe Gly Asn Arg Arg Glu Arg Glu 20 25 6 26 PRT Mus musculus 6 AspLys Gln Thr Leu Leu Gln Asn Glu Gln Leu Tyr Gln Pro Leu Lys 1 5 10 15Asp Arg Glu Tyr Asp Gln Tyr Ser His Leu 20 25 7 26 PRT Mus musculus 7Glu Val Gln Ala Leu Leu Lys Asn Glu Gln Leu Tyr Gln Pro Leu Arg 1 5 1015 Asp Arg Glu Asp Thr Gln Tyr Ser Arg Leu 20 25 8 27 PRT Mus musculus 8Ala Ala Ile Ala Ser Arg Glu Lys Ala Asp Ala Val Tyr Thr Gly Leu 1 5 1015 Asn Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu 20 25 9 27 PRT Musmusculus 9 Glu Leu Glu Ser Lys Lys Val Pro Asp Asp Arg Leu Tyr Glu GluLeu 1 5 10 15 Asn His Val Tyr Ser Pro Ile Tyr Ser Glu Leu 20 25 10 32PRT Homo sapiens 10 Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly TyrMet Thr Leu 1 5 10 15 Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn IleTyr Leu Thr Leu 20 25 30 11 26 PRT Mus musculus 11 Asp Met Pro Asp AspTyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 1 5 10 15 Leu Asp Asp CysSer Met Tyr Glu Asp Ile 20 25 12 26 PRT Homo sapiens 12 Asp Ala Gly AspGlu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 1 5 10 15 Leu Asp AspCys Ser Met Tyr Glu Asp Ile 20 25 13 26 PRT Mus musculus 13 Asp Gly LysAla Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asn 1 5 10 15 Ile AspGln Thr Ala Thr Tyr Glu Asp Ile 20 25 14 26 PRT Homo sapiens 14 Asp SerLys Ala Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asp 1 5 10 15 IleAsp Gln Thr Ala Thr Tyr Glu Asp Ile 20 25 15 26 PRT Gallus gallus 15 AspArg Gln Asn Leu Ile Ala Asn Asp Gln Leu Tyr Gln Pro Leu Gly 1 5 10 15Glu Arg Asn Asp Gly Gln Tyr Ser Gln Leu 20 25 16 27 PRT Bovine leukemiavirus 16 Pro Glu Ile Ser Leu Thr Pro Lys Pro Asp Ser Asp Tyr Gln Ala Leu1 5 10 15 Leu Pro Ser Ala Pro Glu Ile Tyr Ser His Leu 20 25 17 26 PRTBovine leukemia virus 17 Asp Tyr Gln Ala Leu Leu Pro Ser Ala Pro Glu IleTyr Ser His Leu 1 5 10 15 Ser Pro Val Lys Pro Asp Tyr Ile Asn Leu 20 2518 27 PRT Human herpesvirus 4 18 Asp Pro Tyr Trp Gly Asn Gly Asp Arg HisSer Asp Tyr Gln Pro Leu 1 5 10 15 Gly Thr Gln Asp Gln Ser Leu Tyr LeuGly Leu 20 25 19 17 PRT Human herpesvirus 4 19 Met Pro Thr Phe Tyr LeuAla Leu His Gly Gly Gln Thr Tyr His Leu 1 5 10 15 Ile 20 26 PRT Musmusculus 20 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly LeuSer 1 5 10 15 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 20 25 21 26 PRTMus musculus 21 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln GlyLeu Ser 1 5 10 15 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 20 25 22 26PRT Mus musculus 22 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr GlnGly Leu Ser 1 5 10 15 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 20 25 2327 PRT Mus musculus 23 Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro AspTyr Glu Pro Ile 1 5 10 15 Arg Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu 2025 24 27 PRT Mus musculus 24 Asn Lys Glu Arg Pro Pro Pro Val Pro Asn ProAsp Tyr Glu Pro Ile 1 5 10 15 Arg Lys Gly Gln Arg Asp Leu Tyr Ser GlyLeu 20 25 25 27 PRT Mus musculus 25 Asn Lys Glu Arg Pro Pro Pro Val ProAsn Pro Asp Tyr Glu Pro Ile 1 5 10 15 Arg Lys Gly Gln Arg Asp Leu TyrSer Gly Leu 20 25 26 113 PRT Mus musculus 26 Arg Ala Lys Phe Ser Arg SerAla Glu Thr Ala Ala Asn Leu Gln Asp 1 5 10 15 Pro Asn Gln Leu Tyr AsnGlu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Glu Lys LysArg Ala Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Gln Gln Arg Arg Arg AsnPro Gln Glu Gly Val Tyr Asn Ala Leu Gln 50 55 60 Lys Asp Lys Met Ala GluAla Tyr Ser Glu Ile Gly Thr Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly LysGly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp ThrTyr Asp Ala Leu His Met Gln Thr Leu Ala Pro 100 105 110 Arg 27 64 PRTMus musculus 27 Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn LeuGln Lys 1 5 10 15 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr LysGly Glu Arg 20 25 30 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly LeuSer Thr Ala 35 40 45 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr LeuAla Pro Arg 50 55 60 28 26 PRT Mus musculus 28 Arg Ala Lys Phe Ser ArgSer Ala Glu Thr Ala Ala Asn Leu Gln Ala 1 5 10 15 Cys Lys Leu Met GlnThr Leu Ala Pro Arg 20 25 29 53 PRT Artificial sequence 29 Arg Ala LysPhe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Gly 1 5 10 15 Thr LysGly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 20 25 30 Gly LeuSer Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 35 40 45 Thr LeuAla Pro Arg 50 30 113 PRT Artificial sequence 30 Arg Ala Lys Phe Ser ArgSer Ala Glu Thr Ala Ala Asn Leu Gln Asp 1 5 10 15 Pro Asn Gln Leu TyrAsn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Glu LysLys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Gln Gln Arg Arg ArgAsn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln 50 55 60 Lys Asp Lys Met AlaGlu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu 65 70 75 80 Arg Arg Arg GlyLys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys AspThr Glu Asp Ala Leu His Met Gln Thr Leu Ala Pro 100 105 110 Arg 31 113PRT Artificial sequence 31 Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr AlaAla Asn Leu Gln Asp 1 5 10 15 Pro Asn Gln Leu Tyr Asn Glu Leu Asn LeuGly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Glu Lys Lys Arg Ala Arg AspPro Glu Met Gly Gly Lys 35 40 45 Gln Gln Arg Arg Arg Asn Pro Gln Glu GlyVal Tyr Asn Ala Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser GluIle Gly Thr Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp GlyLeu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Phe Asp Ala LeuHis Met Gln Thr Leu Ala Pro 100 105 110 Arg 32 15 PRT Artificialsequence UNSURE (2)..(3) Xaa = any amino acid 32 Tyr Xaa Xaa Leu Xaa XaaXaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Leu 1 5 10 15 33 15 PRT Artificialsequence UNSURE (2)..(3) Xaa = any amino acid 33 Tyr Xaa Xaa Leu Xaa XaaXaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Ile 1 5 10 15

What is claimed is:
 1. A method to identify compounds capable ofregulating actin polymerization in a T lymphocyte, comprising: (a)contacting a putative regulatory compound with a T lymphocyte having a Tcell receptor chain selected from the group consisting of a zeta chainand an epsilon chain, to form a contacted lymphocyte; (b) combining saidcontacted lymphocyte with a molecule capable of inducing thephosphorylation of said zeta chain or said epsilon chain; and (c)assessing the ability of said putative regulatory compound to regulateactin polymerization in said lymphocyte.
 2. The method of claim 1,further comprising assessing the amount of interleukin-2 produced bysaid lymphocyte.
 3. The method of claim 1, wherein said step ofcontacting comprises administering said putative regulatory compound bya technique selected from the group consisting of electroporation,microinjection, cellular expression, use of ion carrying agents and useof detergents for cell permeabilization.
 4. The method of claim 3,wherein said cellular expression is accomplished using an expressionsystem selected from the group consisting of naked nucleic acidmolecules, recombinant virus, retrovirus expression vectors andadenovirus expression vectors.
 5. The method of claim 1, wherein saidmethod further comprises stimulating said lymphocyte, prior to said stepof assessing, with a stimulatory compound selected from the groupconsisting of an antibody specific for a T cell receptor, an antibodyspecific for said ζ chain, an antibody specific for said ε chain,mitogens, lectins and major histocompatibility molecules associated withan antigenic peptide.
 6. The method of claim 1, wherein said methodfurther comprises the step of determining when said lymphocyte hasundergone a morphological change, said change selected from the groupconsisting of changes in adhesion, growth, shape and motilitycharacteristics.
 7. The method of claim 1, wherein said method furthercomprises the step of determining when said lymphocyte has undergone achange in homing activity.
 8. The method of claim 1, further comprisingdetermining the extent of actin association with said ζ chain or said εchain in said lymphocyte by isolating said ζ or ε chain and detectingactin association using an antibody that specifically binds to actin. 9.The method of claim 1, wherein said method further comprises determiningthe modulation of a molecule selected from the group consisting of focaladhesion kinase, paxillin, vinculin, tensin, PI-3Kα, TCR, Syk, Zap-70,Fyn, Shc, IRS-1, Nck, GRB-2, Lck, VAV, GAP, Raf, MEK, MEKK, MAPK, p38,JNKK, JNK, PLA2, JAK1, JAK2, JAK3, Tyk1, Tyk2, STATs, Myc, Jun, Ets-1,Elk-1, CREB, ATF-2, Yes, Hck, Src, CaM Kinase II, S6-K,sphingomyelinase, casein kinase, PKC, PI-3Kγ, SOS, CD45, HCP, Ssp, Syp,PLCγ1, PLCγ2, PLCβ1, PLCβ2, PLCβ3, PLCβ4, PLA2, Grb2, C5aR, IL-8R,MIP1αR, MIP1βR, MCP-1R, MCP-3R, PAFR, FMLPR, LTB₄R, GRPR, and Fasreceptor.
 10. The method of claim 1, wherein said method furthercomprises determining apoptosis of said lymphocytes.
 11. The method ofclaim 1, wherein said T lymphocyte is selected from the group consistingof T cell lines, T cell clones and T cell hybridomas.
 12. The method ofclaim 1, wherein said T cell receptor chain comprises at least oneimmunoreceptor tyrosine-based activation motif.
 13. The method of claim1, wherein said T cell receptor chain comprises an immunoreceptortyrosine-based activation motif selected from the group consisting ofthe amino acid sequence SEQ ID NO:1 and SEQ ID NO:2.
 14. A method toregulate actin polymerization in a T lymphocyte, comprising contacting aT lymphocyte with an effective amount of a regulatory reagent that iscapable of altering the activity of an immunoreceptor tyrosine-basedactivation motif of a ζ chain of a T cell receptor.
 15. The method ofclaim 14, wherein said T lymphocyte is selected from the groupconsisting of a mature and an immature T lymphocyte.
 16. The method ofclaim 14, wherein said activity is altered by a mechanism selected fromthe group consisting of altering the interaction between saidimmunoreceptor tyrosine-based activation motif and its substrate,altering the interaction between said immunoreceptor tyrosine-basedactivation motif and its target molecule and altering the concentrationof said immunoreceptor tyrosine-based activation motif in saidlymphocyte.
 17. The method of claim 14, wherein said method regulates aT lymphocyte function selected from the group consisting of growth,differentiation, homing, proliferation, apoptosis and anergy.
 18. Themethod of claim 14, wherein said method regulates the production ofinterleukin-2.
 19. The method of claim 14, wherein said immunoreceptortyrosine-based activation motif comprises the third immunoreceptortyrosine-based activation motif of a T cell receptor zeta chain.
 20. Themethod of claim 14, wherein said immunoreceptor tyrosine-basedactivation motif comprises the amino acid sequence SEQ ID NO:1.
 21. Themethod of claim 14, wherein said regulatory reagent binds to saidimmunoreceptor tyrosine-based activation motif.
 22. The method of claim14, wherein said regulatory reagent binds to an SH2 domain of a proteinselected from the group consisting of a src-family kinase, a syk-familykinase and an adaptor molecule.
 23. The method of claim 14, wherein saidregulatory reagent is selected from the group consisting of aprotein-based compound, a carbohydrate-based compound, a lipid-basedcompound, a nucleic acid-based compound, a natural organic compound, asynthetically derived organic compound, an antibody and fragmentsthereof.
 24. The method of claim 14, wherein said regulatory reagent isselected from the group consisting of a peptide, a polypeptide and anantibody.
 25. The method of claim 14, wherein said regulatory reagentcomprises an SH2 domain of a protein selected from the group consistingof Fyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck, fak-B, PI-3kinase and 14-3-3.
 26. The method of claim 14, wherein said regulatoryreagent comprises an SH2 domain of a protein selected from the groupconsisting of Fyn, Lck, Zap-70, Shc, Syk, fak-B and 14-3-3.
 27. Themethod of claim 14, wherein said peptide is phosphorylated.
 28. Themethod of claim 14, wherein said regulatory reagent comprises anantibody specific for a protein selected from the group consisting ofFyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck, fak-B, PI-3kinase, 14-3-3, profilin, villin, fragmin, β-actin, gelsolin, depactin,focal adhesion kinase, paxillin, tensin, annexin, ezrin, clathrin-Hchain, vinculin, talin, zixin, profilin, fractinin, cortactin, AFAP-110,p120; β catenin, connexin43 and cadherins.
 29. The method of claim 14,wherein said regulatory reagent is administered by at least one routeselected from the group consisting of oral, nasal, topical, inhaled,transdermal, rectal, intraarticular and parenteral routes.
 30. Themethod of claim 14, wherein said regulatory reagent is administered byat least one route selected from the group consisting of topical, oral,aerosol, intravenous, intraarticular and intramuscular.
 31. The methodof claim 14, wherein said method regulates T lymphocytes involved in adisease selected from the group consisting of an immunoproliferativedisease, immunodeficiency disease, cancer, autoimmune disease,infectious disease, allergic response and graft rejection.
 32. Themethod of claim 14, wherein said effective amount reduces T cellreceptor activation in said lymphocyte when compared with T cellreceptor activation in lymphocytes that have not been contacted withsaid regulatory reagent.
 33. The method of claim 14, wherein saideffective amount alters actin polymerization upon T cell receptorcross-linking as to actin polymerization resulting from T cell receptorcross-linking in the absence of said reagent.
 34. The method of claim14, wherein said effective amount reduces production of interleukin-2 bysaid lymphocyte or induces the death of said lymphocyte.
 35. A method toregulate actin polymerization in a T lymphocyte, comprising contacting aT lymphocyte with an effective amount of a regulatory reagent thatalters the activity of an immunoreceptor tyrosine-based activation motifof an ε chain of a T cell receptor.
 36. The method of claim 35, whereinsaid immunoreceptor tyrosine-based activation motif comprises the aminoacid sequence SEQ ID NO:2.
 37. The method of claim 35, wherein saidregulatory reagent binds to an SH2 domain of a protein selected from thegroup consisting of a src-family kinase, a syk-family kinase and anadaptor molecule.
 38. The method of claim 35, wherein said regulatoryreagent comprises an SH2 domain of a protein selected from the groupconsisting of Fyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck,fak-B, PI-3 kinase and 14-3-3.
 39. The method of claim 35, wherein saideffective amount reduces production of interleukin-2 by said lymphocyteor induces the death of said lymphocyte.
 40. In a cellular system wherea src-family tyrosine kinase is contacted with a T cell receptor chainselected from the group consisting of a ζ chain and an ε chain that isregulated by said src-family tyrosine kinase, the improvement comprisingregulating actin polymerization by contacting a T lymphocyte with areagent capable of binding to a protein selected from the groupconsisting of a third ITAM of a ζ chain, an ITAM of an ε chain and anSH2 domain.
 41. A formulation capable of regulating actin polymerizationin a T lymphocyte, said formulation comprising: (a) a regulatory reagentthat alters the activity of a molecule selected from the groupconsisting of an immunoreceptor tyrosine-based activation motif of a ζchain of a T cell receptor and an immunoreceptor tyrosine-basedactivation motif of a ε chain of a T cell receptor in a cell; and (b) apharmaceutically acceptable carrier.
 42. The formulation of claim 41,wherein said formulation further comprises a compound selected from thegroup consisting of cytochalasins and a molecule that regulates aprotein selected from the group consisting of focal adhesion kinase,paxillin, tensin, annexin, ezrin, clathrin-H chain, vinculin, talin,zixin, profilin, fractinin, cortactin, AFAP-110, p120, β catenin,connexin43 and cadherins.
 43. A kit to identify compounds capable ofregulating actin polymerization in a T lymphocyte, said kit comprising:(a) a cell comprising a T cell receptor chain selected from the groupconsisting of a ζ chain, an ε chain, and actin monomers; and (b) a meansfor detecting the polymerization of said actin monomers.
 44. The kit ofclaim 43, wherein said cell is selected from the group consisting ofJurkat cells, HPB cells, MM-16.11 cells, MM-16.12 cells, MM-16.2 cells,MM-16.5 cells, 17.1 cells, MM-17.2 cells, 122.26 cells, HL-60 cells, H-9cells, peripheral T cells, PBMC Cells, lymph node T cells, splenic Tcells, thymocytes, intraepithelial lymphocytes and tumor infiltratinglymphocytes.
 45. The kit of claim 43, wherein said means for detectingcomprises an antibody that binds specifically to actin.